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標題: 大量表現輔助蛋白及促進細胞循環以提升農桿菌轉殖水稻效率
Increase Agrobacteria-mediated rice transformation efficiency by expression of accessory proteins and starting cell cycle
作者: 郭璧輝
Kuo, Pi-Hui
關鍵字: Agrobacteria;農桿菌;rice;transformation;accessory protein;cell cycle;轉殖;水稻;輔助蛋白;細胞週期
出版社: 農藝學系所
引用: 林家誠. (2010). Increase Agrobacteria-mediated plant transformation efficiency by expression of accessory protein. 國立中興大學生物科技研究所碩士論文. Baulcombe, D. (2004). RNA silencing in plants. Nature 431, 356–363. Gelvin, S.B. (1998). The introduction and expression of transgenes in plants. Curr Opin Biotechnol. 9, 227-232. Gelvin, S.B. (2003). Improving plant genetic engineering by manipulating the host. Trends Biotechnol. 21, 95-98. Godon-Kamm, W., Dilkes, B.P., Lowe, K., Hoerster, G., Sun, X.F., Ross, M., Church, L., Bunde, C., Farrell, J., Hill, P., Maddock, S., Snyder, J., Sykes, L., Li, Z.S., Woo, Y.M., Bidney, D., and Larkins, B.A. (2002). Stimulation of the cell and mayze transformation by disruption of the plant retinoblastoma pathway. Proc Natl Acad Sci USA. 99, 11975-11980. Hwang, H.H., and Gelvin, S.B. (2004). Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. Plant Cell. 16, 3148-3167. Lacroix, B., Vaidya, M., Tzfira, T., and Citovsky, V. (2005). The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation. Embo J. 24, 428-437. Levy, A., Dafny-Yelin, M., and Tzfira, T. (2008). Attackingthe defenders: plant viruses fight back. Trents Microbiol. 16, 194-197. Li, J., Krichevsky, A., Vaidya, M., Tzfira, T., and Citovsky, V. (2005a). Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium. Proc Natl Acad Sci USA. 102, 5733-5738. Li, J., Vaidya, M., White, C., Vainstein, A., Citovsky, V., and Tzfira, T. (2005b). Involvement of KU80 in T-DNA integration in plant cells. Proc Natl Acad Sci USA. 102, 19231-19236. Mysore, K.S., Nam, J., and Gelvin, S.B. (2000). An Arabidopsis histone H2A mutant 20 is deficient in Agrobacterium T-DNA integration. Proc Natl Acad Sci USA. 97, 948-953. Tzfira, T., and Citovsky, V. (2006). Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol. 17, 147-154. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An ehanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949-956. Zhu, Y., Nam, J., Humara, J.M., Mysore, K.S., Lee, L.Y., Cao, H., Valentine, L., Li, J., Kaiser, A.D., Kopecky, A.L., Hwang, H.H., Bhattacharjee, S., Rao, P.K., Tzfira, T., Rajagopal, J., Yi, H., Veena, Yadav, B.S., Crane, Y.M., Lin, K., Larcher, Y., Gelvin, M.J., Knue, M., Ramos, C., Zhao, X., Davis, S.J., Kim, S.I., Ranjith-Kumar, C.T., Choi, Y.J., Hallan, V.K., Chattopadhyay, S., Sui, X., Ziemienowicz, A., Matthysse, A.G., Citovsky, V., Hohn, B., and Gelvin, S.B. (2003). Identification of Arabidopsis rat mutants. Plant Physiol. 132, 494-505.

Agrobacterium-mediated T-DNA transfer is well-known to be a powerful plant transformation tool. This method mostly confer single to low-copies of integrated gene, therefore leads to less problems in gene-silencing. Although many model plants can be transformed by Agrobacterium, a lot of economically important crops are still “recalcitrant” to transformation. We therefore aim to improve plant transformation efficiency using rice as a test system. Accessory proteins, encoded by enhance transformation (ET) genes, are plant factors that involve in T-DNA transfer process within plant cell. By co-transformation of two Agrobacterium horbring two T-DNAs, one carry the ET gene and the other harbor the GUS reporter gene, expression of GUS enzyme was increased in presence of several ET genes, indicating an increase of T-DNA transfer efficiency. In this study, several modifications were employed to optimize the efficiency of ET gene. Firstly, to develop vector that can cohabit with the reporter-carrying vector in the same Agrobacterium, several antibiotics were examined and tetracycline resistance gene was chosen. Secondly, to increase expressions of ET genes, four promoters were tested and 2X35S-U was found to exhibit the strongest activity in rice. Thirdly, to decrease the selection preference of ET gene so to avoid its co-integration with target gene, plant hygromycin selection marker was eliminated from vector that harbors the ET gene. Fourthly, to inhibit gene-silencing effect of plant on the incoming T-DNA, suppressors borrowed from various viruses were screened and P19 was chosen as it confers the highest GUS expressions. Finally, a vector that contains 2X35S-U promoter and P19 viral suppressor, but not the plant selection marker, was constructed to host expressions of various ET genes. In the preliminary test using the constructed vector, BTI exhibited the best effect on enhance transformation. Moreover, a RepA gene that may promote cell cycles, presumably by inhibiting RB protein which blocks G1/S transition, was tested as an ET gene. However, no increase of transformation efficiency was observed.
其他識別: U0005-1608201113484500
Appears in Collections:農藝學系

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